U.S. patent application number 10/740906 was filed with the patent office on 2004-09-09 for precision controlled thermostat for capillary electrophoresis.
Invention is credited to Deka, Chiranjit, Fallon, Joseph M., Karger, Barry L., Miller, Arthur W..
Application Number | 20040173457 10/740906 |
Document ID | / |
Family ID | 32682290 |
Filed Date | 2004-09-09 |
United States Patent
Application |
20040173457 |
Kind Code |
A1 |
Miller, Arthur W. ; et
al. |
September 9, 2004 |
Precision controlled thermostat for capillary electrophoresis
Abstract
A thermostat control system, that can be configured to include
an array of two or more capillary columns or two or more channels
in a microfabricated device, is disclosed. A thermally conductive
material is in contact with each column or channel in the array.
One or more independently controlled heating or cooling elements is
positioned adjacent to or within the thermally conductive material,
each heating or cooling element being connected to a source of
heating or cooling. One or more independently controlled
temperature sensing elements and one or more independently
controlled temperature probes are also positioned adjacent to or
within the thermally conductive material. Each temperature sensing
element is connected to a temperature controller, and each
temperature probe is connected to a thermometer. When the system is
in use, each source of heating or cooling is automatically
regulated by the temperature controller in response to feedback
from one or more of the temperature sensing elements so as to
control temperature stability to within a specified range, and the
temperature controller is automatically regulated in response to
feedback from one or more of the temperature probes to the
thermometer so as to maintain the reference temperature of the
thermally conductive material within a specified range of a pre-set
target temperature.
Inventors: |
Miller, Arthur W.; (Woburn,
MA) ; Deka, Chiranjit; (Andover, MA) ; Fallon,
Joseph M.; (Wakefield, MA) ; Karger, Barry L.;
(Newton, MA) |
Correspondence
Address: |
WEINGARTEN, SCHURGIN, GAGNEBIN & LEBOVICI LLP
TEN POST OFFICE SQUARE
BOSTON
MA
02109
US
|
Family ID: |
32682290 |
Appl. No.: |
10/740906 |
Filed: |
December 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60435885 |
Dec 20, 2002 |
|
|
|
Current U.S.
Class: |
204/451 ;
204/602 |
Current CPC
Class: |
G05D 23/1931 20130101;
G05D 23/24 20130101; G01N 27/44708 20130101; G01N 27/44782
20130101 |
Class at
Publication: |
204/451 ;
204/602 |
International
Class: |
C25B 007/00; G01N
027/26 |
Claims
What is claimed is:
1. A thermostat control system comprising: one or more capillary
columns or one or more channels in a microfabricated device; a
thermally conductive material in contact with said capillary
columns or channels; one or more independently controlled heating
or cooling elements positioned adjacent to or within said thermally
conductive material, wherein each heating or cooling element is
connected to a source of heating or cooling; one or more
independently controlled temperature sensing elements positioned
adjacent to or within said thermally conductive material, wherein
each temperature sensing element is connected to a temperature
controller; and one or more independently controlled temperature
probes positioned adjacent to or within said thermally conductive
material, wherein each temperature probe is connected to a
thermometer and wherein, when said system is in use, each said
source of heating or cooling is automatically regulated by said
temperature controller in response to feedback from one or more of
said temperature sensing elements so as to control temperature
stability to within a specified range, and said temperature
controller is automatically regulated in response to feedback from
one or more of said temperature probes to said thermometer so as to
maintain the reference temperature of said thermally conductive
material within a specified range of a pre-set target
temperature.
2. The control system of claim 1, said system comprising multiple
independent said capillary columns or multiple independent said
channels in a microfabricated device, or multiple independent
clusters of said capillary columns or said channels, wherein said
columns or channels are distributed among different
instruments.
3. A thermostat control system comprising: one or more capillary
columns; a capillary body support surrounding each said capillary
column; two or more discs of a first thermally conductive material
supporting said capillary body support or supports; a second
thermally conductive material surrounding said capillary body
support or supports adjacent to said discs of said first thermally
conductive material, wherein the melt temperature of said first
thermally conductive material is higher than the melt temperature
of said second thermally conductive material; one or more
independently controlled heating or cooling elements positioned
adjacent to or within said first and second thermally conductive
materials, wherein each heating or cooling element is connected to
a source of heating or cooling; and one or more independently
controlled temperature sensing elements positioned adjacent to or
within said first and second thermally conductive materials,
wherein each temperature sensing element is connected to a
temperature controller.
4. The control system of claim 3, further comprising: one or more
independently controlled temperature probes positioned adjacent to
or within said first and second thermally conductive materials,
wherein each temperature probe is connected to a thermometer and
wherein, when said system is in use, each said source of heating or
cooling is automatically regulated by said temperature controller
in response to feedback from one or more of said temperature
sensing elements so as to control temperature stability to within a
specified range and said temperature controller is automatically
regulated in response to feedback from one or more of said
temperature probes to said said thermometer so as to maintain the
reference temperature of said first and second thermally conductive
materials within a specified range of a pre-set target
temperature.
5. The control system of claim 4, said system comprising multiple
independent said capillary columns, or multiple independent
clusters of said capillary columns, wherein said columns or
channels are distributed among different instruments.
6. A thermostat array control system comprising: two or more
capillary columns or two or more channels in a microfabricated
device, wherein said two or more columns or said two or more
channels are associated in an array; a thermally conductive
material in contact with said capillary columns or channels; one or
more independently controlled heating or cooling elements
positioned adjacent to or within said thermally conductive
material, wherein each heating or cooling element is connected to a
source of heating or cooling; one or more independently controlled
temperature sensing elements positioned adjacent to or within said
thermally conductive material, wherein each temperature sensing
element is connected to a temperature controller; and one or more
independently controlled temperature probes positioned adjacent to
or within said thermally conductive material, wherein each
temperature probe is connected to a thermometer and wherein, when
said system is in use, each said source of heating or cooling is
automatically regulated by said temperature controller in response
to feedback from one or more of said temperature sensing elements
so as to control temperature stability to within a specified range,
and said temperature controller is automatically regulated by
feedback from one or more of said temperature probes to said said
thermometer so as to maintain the reference temperature of said
thermally conductive material within a specified range of a pre-set
target temperature.
7. A thermostat array control system comprising: two or more
capillary columns, wherein said two or more columns are associated
in an array; a capillary body support surrounding each said
capillary column; two or more discs of a first thermally conductive
material supporting said capillary body supports; a second
thermally conductive material surrounding said capillary body
supports adjacent to said discs of said first thermally conductive
material; one or more independently controlled heating or cooling
elements positioned adjacent to or within said first and second
thermally conductive materials, wherein each heating or cooling
element is connected to a source of heating or cooling; and one or
more independently controlled temperature sensing elements
positioned adjacent to or within said first and second thermally
conductive materials, wherein each temperature sensing element is
connected to a temperature controller.
8. The control system of claim 7, further comprising: one or more
independently controlled temperature probes positioned adjacent to
or within said first and second thermally conductive materials,
wherein each temperature probe is connected to a thermometer and
wherein, when said system is in use, each said source of heating or
cooling is automatically regulated by said temperature controller
in response to feedback from one or more of said temperature
sensing elements so as to control temperature stability to within a
specified range and said temperature controller is automatically
regulated in response to feedback from one or more of said
temperature probes to said said thermometer so as to maintain the
reference temperature of said first and second thermally conductive
materials within a specified range of a pre-set target
temperature.
9. The control system of claim 6, wherein said one or more heating
or cooling elements are positioned adjacent to individual said
columns or channels and wherein each individual said column or
channel is thermally insulated from every other said column or
channel.
10. The control system of claim 6, said system further comprising
multiple said columns or channels, wherein two or more of said
multiple columns or channels are heated or cooled by a single
heating or cooling element and multiple clusters of such columns or
channels heated or cooled by a single heating or cooling element
are associated in said thermostat array control system and wherein
said columns or channels heated or cooled by a single heating or
cooling element within a cluster of said columns or channels can be
maintained at the same temperature and different clusters within
said array are independently controllable.
11. The control system of claim 7, wherein said one or more heating
or cooling elements are positioned adjacent to individual said
columns and wherein each individual said column is thermally
insulated from every other said column.
12. The control system of claim 7, said system further comprising
multiple said columns, wherein two or more of said multiple columns
are heated or cooled by a single heating or cooling element and
multiple clusters of such columns heated or cooled by a single
heating or cooling element are associated in said thermostat array
control system and wherein said columns heated or cooled by a
single heating or cooling element within a cluster of said columns
can be maintained at the same temperature and different clusters
within said array are independently controllable.
13. The control system of claim 6 or claim 7, wherein said one or
more heating or cooling elements are also used as said temperature
sensing elements.
14. The control system of claim 6, wherein said capillary columns
or channels are suitable for use in a separation method calling for
an electric field and said columns or channels are electrically
isolated from said heating or cooling elements.
15. The control system of claim 7, wherein said capillary columns
are suitable for use in a separation method calling for an electric
field and said columns are electrically isolated from said heating
or cooling elements.
16. The control system of claim 6, wherein said heating or cooling
elements surround said capillary columns or channels.
17. The control system of claim 7, wherein said heating or cooling
elements surround said capillary columns or channels.
18. The control system of claim 6, comprising two or more
independently controlled heating or cooling elements associated
with an individual said column or channel, wherein said two or more
heating or cooling elements are positioned along said associated
column or channel so as to be capable of inducing a thermal
gradient along the length of said column or channel.
19. The control system of claim 7, comprising two or more
independently controlled heating or cooling elements associated
with an individual said column, wherein said two or more heating or
cooling elements are positioned along said associated column so as
to be capable of inducing a thermal gradient along the length of
said column.
20. The control system of claim 6, wherein said independently
controlled heating or cooling elements associated with an
individual said column or channel are configured for temperature
programming.
21. The control system of claim 7, wherein said independently
controlled heating or cooling elements associated with an
individual said column are configured for temperature
programming.
22. The control system of claim 6 or claim 7, wherein said heating
or cooling elements are solid-state.
23. The control system of claim 6 or claim 7, wherein said heating
or cooling elements are a fluid.
24. The control system of claim 23, wherein said fluid heating or
cooling element is a liquid.
25. The control system of claim 23, wherein said fluid heating or
cooling element is a gas.
26. A method of finding the optimum temperature for an analysis
procedure for a particular sample, said method comprising the steps
of: providing the thermostat array control system of claim 6;
determining the number of different temperature values to be
examined; including within the thermostat array a number of columns
or channels equal to the number of different temperature values to
be examined; configuring each said column or channel for carrying
out said analysis procedure for said sample; adjusting said
temperature controller associated with said thermostat array so as
to maintain the temperature at each individual said column or
channel at one of said different temperature values to be examined;
carrying out said analysis procedure on different aliquots of said
sample simultaneously in each of said columns or channels, each of
said individual columns or channels being maintained at a different
one of said temperature values to be examined; and comparing the
results of said analysis procedure carried out in said individual
columns or channels to determine the optimum temperature for said
analysis procedure for said sample.
27. The method of claim 26, wherein said analysis procedure is
constant denaturant capillary electrophoresis.
28. The method of claim 26, wherein said analysis procedure is a
single strand conformational polymorphism analysis.
29. A method of carrying out an analysis procedure simultaneously
for multiple samples, each said sample having a different
temperature optimum for said procedure, said method comprising the
steps of: providing the thermostat array control system of claim 6;
determining the number of different samples to be examined;
including within the thermostat array a number of columns or
channels, or clusters of columns or channels, equal to the number
of different samples to be examined; configuring each said column
or channel for carrying out said analysis procedure for one of said
multiple samples; adjusting said temperature controller associated
with said thermostat array so as to maintain the temperature at
each individual said column or channel at the optimum temperature
for carrying out said analysis procedure for an individual said
sample; carrying out said analysis procedure on said different
samples simultaneously in each of said columns or channels, each of
said individual columns or channels being maintained at the optimum
analysis temperature for the individual said sample associated with
said individual column; and obtaining the results of said analysis
procedure for each of said samples.
30. The method of claim 29, wherein said analysis procedure is
constant denaturant capillary electrophoresis.
31. The method of claim 29, wherein said analysis procedure is a
single strand conformational polymorphism analysis.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority of U.S. Provisional
Application No. 60/435,885 filed Dec. 20, 2002 entitled, PRECISION
CONTROLLED THERMOSTAT FOR CAPILLARY ELECTROPHORESIS, the whole of
which is hereby incorporated by reference herein.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] N/A
BACKGROUND OF THE INVENTION
[0003] Capillary electrophoresis is a powerful technique used to
separate molecules based on size and/or charge. In the analysis of
samples requiring identical (or similar) separation conditions, it
frequently is useful to have all of the columns of an array, e.g.
all of the separation columns of a DNA sequencer or analyzer, held
at the same temperature. On the other hand, there are many
situations where the possibility of adjusting the temperature
individually for each separation element of an array, or for
clusters of separation elements within an array, would be of great
practical importance. For example, an array of ten capillary
columns, operating at ten different temperatures, could be used to
find an optimum temperature for separation of certain specific
species of DNA molecules. Conversely, ten different samples, each
requiring a different temperature for optimum analysis, could be
run in parallel with a similar increase in productivity beyond what
individual runs carried out consecutively would achieve.
[0004] Foret et al., U.S. patent application Ser. No. 09/979,622,
incorporated by reference herein, describes an embodiment of a
thermostat array comprising essentially a number of heaters, each
consisting of a cylindrical volume of thermally conductive material
surrounded by an electrically powered heating element, whose power
is adjusted in a feedback loop using an electrical temperature
sensor such as an RTD, thermocouple, or thermistor. Although a
single feedback system such as this can be used to maintain a
thermostat setting with a high degree of stability
(.+-.0.01.degree. C. to .+-.0.02.degree. C.), it is extremely
difficult to reset the temperature with this same degree of
accuracy. "Resettability" is defined as the ability to set any
given temperature (e.g., between 0.degree. C.-150.degree. C.) and
achieve the same temperature every time, and for every heater.
Because resettability to this level demands reference to some
calibration standard, of which the most convenient is for absolute
accuracy of temperature, the resettability requirements discussed
herein are in practice usually absolute temperature accuracy
requirements as well.
[0005] The need for a stringent resettability tolerance can arise
in applications such as Constant Denaturant Capillary
Electrophoresis (CDCE), in which DNA fragments are separated on the
basis of minute differences in melting temperature. Under certain
CDCE protocols, it is critical that the migration time differences
between peaks be highly uniform, to obtain a high degree of
automation of peak detection, and of high-confidence peak area
measurement and matching of peaks and other features among
electropherograms from different capillaries. Differences in
separation temperature of only a few hundredths of a degree can
shift peaks enough to make this infeasible, especially when peaks
are small. (In general, whenever the signal-to-noise ratio is low,
small peaks can be partly or fully masked by nearby larger peaks,
Shifting of peaks can compound the likelihood of such events.)
Therefore, while it may be adequate to set the CDCE run temperature
only to a resolution of 0.1.degree. C., the resettability of that
temperature in these cases should be at least to a few hundredths
of a degree, and preferably no more than .+-.0.01-0.02.degree.
C.
[0006] A fundamental cause for the difficulty in achieving
resettability in such a system is the intrinsic inaccuracy of the
various electronic components of the control system, which results
from the materials and design of the components, and which can also
be dependent on the ambient temperature of the electronic elements
and on other factors. A particular example of this is the unit to
unit variations in the performance of individual sensors. For
example, assuming a sensor rated for stability of temperature
response to within .+-.0.01.degree. C., there might be unit-to-unit
variation of the temperature response of .+-.0.1.degree. C. or
more. (This is error solely from the sensing element(s). The
electronic measurement system for the sensor commonly contributes a
large additional error.) This variance, often referred to as the
"interchangeability tolerance," defines how far a specific sensor
within that group may vary from the nominal "Temperature-Response
Curve." Using such sensors, one could obtain a temperature
stability of .+-.0.01.degree. C., but the resettability or absolute
temperature accuracy cannot be better than .+-.0.1.degree. C. Thus,
there exists a need in the art to obtain a practical solution
whereby one can achieve a high degree of both stability and
resettability simultaneously. Though this discussion uses a very
stringent tolerance of .+-.0.01.degree. C. for purposes of
illustration, the same arguments apply for other values, e.g., when
the required stability and resettability is .+-.0.1.degree. C., but
the unit-to-unit variation and other factors, including those
discussed-below, lead to worse resettability than .+-.0.1.degree.
C.
[0007] One possible means by which the performance limitation just
described can be overcome is to always use sensors (e.g.,
thermistors) in each heater that are specified to have an
"interchangeability tolerance" of less than .+-.0.01.degree. C. and
to stabilize the ambient temperature for the electronics to a
pre-specified temperature. Although theoretically possible, this
solution is impractical for many industrial applications. For
example, it is difficult to obtain commercially large numbers of
thermistors that are pre-selected to meet an "interchangeability
tolerance" of less than .+-.0.01.degree. C. Therefore, in the
thermostat array described by Foret et al., if one were to utilize
commercial thermistors to control each heater, one would be
required to calibrate each and every control thermistor
individually and use a different set of calibration curves for
temperature controls every time a new set of heaters is used for
measurements. For multiple instruments with multiple heaters, this
requirement places stringent demands on the system that require the
tracking of each heater at all times. This is not only inconvenient
but it also carries the risk of inadvertent but serious mistakes
creeping into the overall process.
[0008] Even if one were to implement such a calibration and
tracking mechanism, the problems caused due to variations in the
electronic components still remain. As a result, even with all the
tracking and calibration of individual thermistors, one may not get
repeatable temperatures unless the ambient temperature of the
control electronics itself is also thermally stabilized, or
provided with highly accurate internal temperature measurement and
subjected to extensive calibration. Such a requirement renders the
system very complex and expensive to build. Thus, a better approach
is still desirable.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention provides a method for overcoming these
problems and provides a thermostat system that permits both
stability and resettability of temperature to within less than
.+-.0.02.degree. C., using commercially available sensors and using
a design that does not require the electronics to be maintained in
any thermally stabilized environment.
[0010] In one embodiment, the invention is directed to a thermostat
control system that can be configured to include one or more
capillary columns or one or more channels in a microfabricated
device. Individual columns or channels, or clusters of columns or
channels can, preferentially, be associated in arrays. A thermally
conductive material is in contact with each column or channel. One
or more independently controlled heating or cooling elements is
positioned adjacent to or within the thermally conductive material,
each heating or cooling element being connected to a source of
heating or cooling. One or more independently controlled
temperature sensing elements and one or more independently
controlled temperature probes are also positioned adjacent to or
within the thermally conductive material. Each temperature sensing
element is connected to a temperature controller, and each
temperature probe is connected to a reference thermometer. When the
system is in use, each source of heating or cooling is
automatically regulated by the temperature controller in response
to feedback from one or more of the temperature sensing elements so
as to control temperature stability to within a specified range,
and the temperature controller is automatically regulated in
response to feedback from one or more of the temperature probes to
the thermometer so as to maintain the reference temperature of the
thermally conductive material within a specified range of a pre-set
target temperature.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0011] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof and from the claims, taken in conjunction with
the accompanying drawings, in which:
[0012] FIGS. 1a-1c show one embodiment of a cluster of individual
capillary columns with associated temperature control and
monitoring elements suitable for use in the thermostat array
control system according to the invention. FIGS. 1a and 1b are an
axial section view and a cross-axial section view, respectively, of
the column cluster, and FIG. 1c is a schematic drawing of the
temperature control and monitoring elements of this embodiment;
[0013] FIGS. 2a and 2b shows another embodiment of a cluster of
individual capillary columns for use in the thermostat array
control system according to the invention;
[0014] FIG. 3 is a schematic drawing of the temperature control
system for an array of six heaters suitable for use in the
thermostat array control system according to the invention;
[0015] FIG. 4 is a schematic drawing showing an embodiment of
distributed temperature control according to the invention, in
which four heaters of the present invention are incorporated in
four different single capillary electrophoresis instrument;
[0016] FIG. 5 shows an embodiment of the thermostat array control
system according to the invention integrated on a microfabricated
device;
[0017] FIG. 6 shows an example of the use of the thermostat array
control system according to the invention for CDCE analysis;
[0018] FIG. 7 shows the results of six heaters maintained at six
different temperatures within less than .+-.0.02.degree. C. of
their respective set temperatures using the thermostat array
control system of the invention;
[0019] FIGS. 8a and 8b are graphs showing the results from the use
of the thermostat array control system according to the invention
for optimization of CDCE separation of a PCR amplified DNA sample;
and
[0020] FIGS. 9a-9c are graphs showing reproducible CDCE separation
using the thermostat array control system according to the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Described herein are individual columns and arrays of
independently controlled thermostat systems according to the
invention useful, e.g., for column separations. Individual
thermostat control systems in an array can be associated with
individual capillary columns (or channels in a microfabricated
device) or with groups (clusters) of such columns or channels. An
array of independently controlled thermostats according to the
invention is useful, e.g., in constant denaturant capillary
electrophoresis as described in K. Khrapko et al., Constant
Denaturant Capillary Electrophoresis (CDCE): A High Resolution
Approach to Mutational Analysis. Nucl. Acid. Res., 22, 1994,
364-269. In CDCE, DNA fragments, for example, are analyzed based on
differences in melting temperature. Specific embodiments of
individual clusters of capillary columns with associated
individually controlled thermostats suitable for use in the
thermostat array control system of the invention are shown in FIGS.
1a-1c and FIGS. 2a-2b. There are many other embodiments that can be
derived from those described herein, which are suitable for
alternative applications, as will be obvious for one skilled in the
art. For example, a temporal gradient may be repeated in a cyclic
manner by ramping the temperature up and down during the
separation, such as is done in cycling temperature capillary
electrophoresis (CTCE), a technique described in Minarik et al.,
Cycling gradient capillary electrophoresis: a low-cost tool for
high-throughput analysis of genetic variations, Electrophoresis
2003, 24, 1716-1722. Simpler nonrepeating gradients and a wide
variety of temperature programming methods would also constitute
suitable applications; e.g., the methods discussed in Li et al.,
Integrated platform for detection of DNA sequence variants using
capillary array electrophoresis, Electrophoresis 2002, 23,
1499-1511. While the focus here is on CDCE and capillaries, the
current invention could equally well apply to other techniques,
including ones not involving electrophoresis, in which much wider
bore tubing is employed (e.g., several millimeters).
[0022] In one embodiment, as shown in FIGS. 1a and 1b, the
solid-state heater component 10 of the thermostat system according
to the invention comprises a cylindrical block 12 of a thermally
conductive material such as copper, brass, or stainless steel,
about 6 inches long and 1 inch in diameter. Hollow channels 14
formed by drilling through the solid cylindrical block 12 run
through the thermally conductive material parallel to the axis of
this cylinder. One or more temperature sensors 16 (e.g.,
thermistors) are embedded on shallow grooves on the surface of the
cylindrical body. Stainless steel capillary tubes 18, inserted
through hollow channels 14 in the cylindrical body, are held in
place by filling up the space between the outer surface of each
tube and the inner surface of each channel, e.g., with a thermal
epoxy 20. The cylindrical outer surface of thermally conductive
block 12 is wrapped with a flexible heating element 22 then covered
further with a layer of insulating foam 24 and protected by a heat
shrink tube 26.
[0023] An advantageous feature in this embodiment is the presence
of multiple capillaries through the heater. With, e.g., four
capillaries, as more clearly shown in FIG. 1b, experiments can be
designed such that a target DNA sequence can be analyzed completely
within one CDCE run, using a separate capillary for a pooled
population of interest, a pooled control population, a positive
control and a negative control. However, the number of capillaries
per heater is not a limitation, and any number of capillaries can
be incorporated in an individual heater. Furthermore, multiple
capillaries in a single heater might be employed differently from
the manner just detailed under other circumstances. Also, as
described subsequently, groups of such heaters can be used to
analyze different DNA targets (requiring different separation
temperatures) simultaneously.
[0024] As shown in schematic form in FIG. 1c, temperature
controller 28, connected to heating element 22, provides a current
to the heating element that is continuously adjusted to maintain a
stable temperature in response to the continuous feedback input it
receives from the sensor 16. Referring also to FIG. 1a, a precision
temperature probe 30, e.g., a thermistor encased in a stainless
steel tube, about 3 inches in length and 0.125 inches in diameter,
in which the thermistor is embedded using thermally conductive
epoxy, is inserted in a hole 32 and used to monitor the absolute
temperature within the thermally conductive block 12 of the heater
body. This temperature probe 30 is connected to a digital
thermometer 34, which provides a feedback to a computer 36
containing an analog output board 38 (D/A board), which is
connected to the temperature controller 28. The analog output board
38 is used to adjust the primary operating reference voltage of the
temperature controller to match the target temperature to within a
desired range.
[0025] It should be noted that wherever heating elements are
referenced herein, it would be possible to add cooling elements as
well for greater temperature control capability. Heating and
cooling elements might also be combined into a single
heating/cooling element, such as a Peltier device. It should also
be noted that temperature sensors and temperatures probes are the
same category of device (temperature transducers), and that the two
terms are used herein only for clarity in distinguishing the two
levels of temperature control in the system according to the
invention. Temperature sensors and probes may be thermisters,
thermocouples, RTDs, PRTs, SPRTs, ICs (semiconductor devices),
infrared detectors, reversible temperature indicating labels, or
any material or device in which some measurable property changes in
a fashion that can be correlated to temperature. Such properties
include resistance, output current, visible color, and infrared
light emission.
[0026] While the preferred embodiment described herein employs an
analog controller, this unit may be substituted in other
embodiments by other types of controllers. As one example, the
control logic (such as on-off control, proportional control, PID
control, fuzzy logic control, or combinations of these in
single-loop or multiloop fashion) may reside in software on a
computer, which through additional hardware such as a solid-state
relay and an external power supply, pulses current to a
heating/cooling element.
[0027] In another embodiment, as shown in FIGS. 2a-2b, the
solid-state heater component of the thermostat comprises a
cylindrical body 40 comprising a first thermally conductive
material 40a, which is cast around four stainless steel capillaries
18 held in place by a number of parallel circular discs 40b of a
second thermally conductive material. One or more temperature
sensors 16 (e.g., thermistors) are embedded on shallow grooves on
the surface of the thermally conductive cylindrical body 40. The
cylindrical outer surface of the thermally conductive block 40 is
wrapped with a flexible heating element 22, then covered further
with a layer of insulating foam 24, and protected by a heat shrink
tube 26. The temperature control mechanism is the same as described
for the embodiment above (FIG. 1c).
[0028] Temperature control of an array of six heaters is shown in
FIG. 3. In this embodiment, an independent reference voltage is
sent by the D/A board 42 to each analog temperature controller 44.
The controller then monitors a voltage from a thermistor embedded
in a heater 46, which is a measure of the temperature of the
thermistor. The controller continuously adjusts the current
supplied to the heater until the voltage from the thermistor is the
same as the reference voltage. The thermometer 48 monitors
temperature concurrently temperature probes 50 and reports its
measurements to the control software 52. At frequent intervals, the
software checks whether the heater temperature has been stable
within a preset range for a predetermined time interval. If this is
true, a test is performed to establish whether this stable
temperature is within tolerance of the target temperature. If the
difference from the target temperature exceeds the tolerance, a
correction to the reference voltage for that heater is computed,
and the D/A board is set to send the corrected voltage on the
appropriate output channel. The process of waiting for heater
temperature to stabilize, testing the temperature against a
tolerance and correcting the reference voltage is repeated
indefinitely.
[0029] While in principle it would be possible to substitute the
controlling temperature sensors with the much more highly accurate
calibrated thermometer and attached probes, and thus avoid the
extra control loop, there are practical difficulties with such an
approach. One is that the rapid responses to temperature
disturbances required demand rapid temperature measurements, but
rapid measurements come at the cost of degrading accuracy. Another
problem is that for best control, the controlling sensors should be
located very close to the heating/cooling element, and are
therefore typically embedded in the apparatus and difficult to
easily replace, reuse, or recalibrate. Difficulties with tracking
logistics have already been remarked upon. In the current
invention, a single multichannel thermometer can be used to provide
highly accurate temperature for many different
temperature-controlled zones, using probes that can easily be
removed from either the thermometer or from the zone being
controlled. This reduces the number of expensive, high-tolerance
components needed to control multiple heaters. Because the same
thermometer and probes can be used over time for many different
sets of heaters, tracking problems are reduced as well.
[0030] FIG. 4 illustrates an embodiment of distributed temperature
control, in which four heaters 54 of the present invention are
incorporated in four different single capillary electrophoresis
instruments 56 (which in principle could instead be four different
multicapillary instruments). Each heater is separately controlled
by a separate analog temperature controller 58 within the
temperature control system of the invention, and the instruments
are operated independently. All the analog controllers are
physically positioned at a central location, similarly to the
fashion illustrated in FIG. 3. A single multichannel digital
thermometer is at that central location as well and is used to
provide the second level of control for all the instruments. Leads
for sensors, probes, and heater power are run from the central
position to the different instruments.
[0031] The examples described above show a thermostat system
suitable for an array of discrete capillaries. Precise and
resettable independent control of temperature is also important in
microfabricated devices. The entire thermostat/capillary column
array described here, including heaters and sensors (thermistors,
RTDs, etc.) and, if needed, also the controllers, can be integrated
on a microfabricated device, e.g., a microchip. Due to the small
size of microchips and the good thermal conductivity of most
substrate materials used for fabrication, e.g., fused silica, the
closely neighboring heated/cooled areas of the thermostat array
could strongly influence each other. To prevent this type of
thermal communication, microdevices implemented with the thermostat
array of the invention need to be equipped with heat insulating
regions between individual temperature controlled channels or
clusters of channels. An example of such a microdevice is depicted
in FIG. 5.
[0032] Referring to FIG. 5, planar microchip 60, having a fused
silica chip body 62, contains multiple channels 64, each associated
with a heating/cooling element 66. Wires 68 connect heating/cooling
elements 66 to individual temperature controllers 72. Temperature
sensors 70 provide feedback to temperature controllers 72, and
temperature probes 76 are connected to reference thermometer 78,
which provides feedback to computer 80, as described above.
Temperature control is as given for previous embodiments.
[0033] To eliminate heat transfer between individual
channel/heating element combinations, through cuts 74 are made
between the channels. The cuts can be further filled with an
insulating material such as polyurethane or polystyrene foam.
Heating elements 66 can be attached from the top and/or the bottom
of the microchip. In addition, the vertical walls of cuts 74 could
be coated with a conductive material and connected to the current
source so as to provide a source of heating/cooling surrounding a
desired channel.
[0034] The temperature sensors 70 and probes 76 (Pt, thermistors,
or other) can be attached from either side of a channel 64.
Alternatively, the heating element itself can serve as the
temperature sensing element if it is made from a material that
changes resistance over time. For example, a conductive (Pt, Cr,
Au, conductive plastic) layer can be deposited directly on the
surface of the microdevice (or inside before the layers of the
device are bonded) by using sputtering or chemical vapor deposition
techniques. Similarly to the earlier described configuration for
capillary column thermostat arrays, multiple channels could also be
heated (cooled) by a single heating/cooling element, and clusters
of such channels could be associated in a thermostat array of the
invention wherein different clusters within the array are
independently controlled.
[0035] An example of the use of the thermostat array of the
invention in a system for CDCE analysis is shown in FIG. 6.
[0036] Referring to FIG. 6, solid state thermostat array 82
includes separation capillaries 84 for CDCE analysis, e.g., of
separate mitochondrial DNA samples. Samples are injected into
individual capillaries 84. The capillaries are also positioned for
comprehensive collection of zones exiting the capillaries. Laser
illumination system 86 produces two point illumination for, e.g.,
laser induced fluorescence (LIF) detection using a spectrograph/CCD
detector 88. Temperature control is as shown in FIG. 5. In this
particular design, the thermostats are used to maintain a constant
temperature in each separation capillary (a different temperature
in each column) to achieve the desired resolution of the DNA
fragments, which are consecutively subjected to LIF velocity
measurement and fraction collection.
[0037] FIG. 7 shows temperature readings over an hour for six
heaters set for six different temperatures using the control system
of the invention. Temperatures are shown to be maintained within
.+-.0.01.degree. C. of their respective set temperatures, based on
the specifications of thermometer employed.
[0038] The following examples are presented to illustrate the
advantages of the present invention and to assist one of ordinary
skill in making and using the same. These examples are not intended
in any way otherwise to limit the scope of the disclosure.
EXAMPLE 1
CDCE Temperature Optimization.
[0039] A thermostat array containing six heaters, as described
herein, was incorporated in a modified DNA sequencer (based on
Spectrumedix 2410) for mutation discovery in pooled populations
using CDCE. For CDCE of a PCR amplified DNA sample, ideally one
might set the CDCE temperature such that for each target sequence
there would be four distinct and well resolved peaks when a
mutation is present (the wildtype homoduplex, the mutant homoduplex
and two wildtype-mutant heteroduplexes). This temperature is
usually close to but not exactly equal to the theoretical melting
temperature (Tm) calculated for the wildtype homoduplex. To
determine the optimum CDCE temperature, an initial CDCE experiment
is conducted on the sample in which the six heaters in the
thermostat array in the instrument are set at six slightly
different temperatures in a narrow range around the calculated
Tm.
[0040] FIG. 8a illustrates such a temperature optimization for a
DNA fragment denoted as CTLA-4E1. The same CTLA-4E1 sample was
injected into one capillary in each of the six heaters, where each
heater was set to a different temperature in the range 73.5.degree.
C. to 77.5.degree. C. At 73.5.degree. C., only a single peak was
observed for the target sequence, representing the case in which
all species of the DNA molecules shown under this peak are in the
unmelted state. At 74.5.degree. C., this peak split into two
distinct peaks, indicating that the one of the heteroduplex species
is already partially melted while the wildtype homoduplex, mutant
homoduplex, and the other heteroduplex are all in a mostly unmelted
state. At 75.5.degree. C., four distinct, well resolved peaks were
observed, which indicates maximum differential melting conditions
between the four species. At 76.0.degree. C., the mutant homoduplex
peak is separated further from the wildtype, but at the same time,
the resolution between the three mutant peaks started deteriorating
significantly. At 76.5.degree. C., the mutant peaks coalesced and
the wildtype peaks got closer to the mutant peak. This represents
the condition in which the mutant homoduplex and two heteroduplexes
are all nearly completely melted and the wildtype partially melted.
Finally, at 77.5.degree. C., all four peaks coalesced into a single
peak, representing the case in which the low-melting domains of all
the four species were completed melted with only the high-melting
domain remaining intact to hold the partially opened
double-stranded DNA fragment together. From this example, one would
conclude that 75.5.degree. C. was the optimum CDCE temperature.
[0041] As can be seen, quite different results are obtained with
even half a degree of temperature difference. FIG. 8b shows
significant variations in migration time and peak resolution with
only 0.1.degree. C. difference in temperature between capillaries.
These results emphasize the importance of maintaining a specific
temperature during such an analysis, as is possible using the
system of the invention.
EXAMPLE 2
Separation Reproducibility in a Modified SpectruMedix DNA
Sequencer.
[0042] Example 1 demonstrated the importance of accurate
temperature settings for CDCE. The present experiment demonstrates
the ability of the heater array to produce the same temperature
environment for each column exactly as set, and to generate
reproducible CDCE separation. Variations in the migration time of
the four peaks were used as measures of this reproducibility. For
this experiment, a CTLA-4E1 sample was injected into all the 24
capillaries and run at the optimum CDCE temperature of 75.5.degree.
C. The resulting electropherograms, aligned to the peak occurring
just before time point 1200, are shown in FIG. 9a. The four major
peaks starting with this alignment peak represent the homoduplex
and heteroduplex species for this sample. For analytical purposes,
a key metric is the migration time differences between pairs of
these peaks. Comparison with FIGS. 8a and 8b shows that the
migration time differences shown here reflect very uniform
temperature between capillaries and heaters.
EXAMPLE 3
Separation Reproducibility in a Modified Beckman Coulter DNA
Sequencer.
[0043] In this experiment, two heaters of the present invention
were incorporated in a commercial DNA sequencer (CEQ 2000, Beckman
Coulter Inc.). A modified 8-capillary array was made by passing 4
capillaries through the first heater and the remaining 4
capillaries through a second heater. The temperature control system
of the present invention was connected directly to the two heaters.
CDCE separation was conducted on identical samples through each of
the 8 capillaries in the array. Temperature had previously been
optimized so that the homoduplex peaks migrated at nearly the same
position, but the heteroduplex peaks migrated significantly slower.
FIG. 9b shows the results after alignment to the leading homoduplex
peaks. The heteroduplex peaks (just before and just after 54
minutes) migrate at a distance from the homoduplexes that is
constant across capillaries and heaters to within a single peak
width.
EXAMPLE 4
Resettability
[0044] This experiment demonstrates the ability of the heaters of
the present invention to reset to exactly the same temperatures in
different days. FIG. 9c shows the resettability of temperatures in
the heaters according to the invention. On two different days, an
identical sample was injected in two capillaries of the modified
SpectruMedix sequencer used in Example 1. Again referencing FIG. 8a
and 8b for the effect of small temperature changes on separation,
the high uniformity of migration times in FIG. 9c demonstrates,
even without aligning the electropherograms to a common peak, that
a very similar temperature was achieved both between capillaries
and between days. The resettability possible with the system of the
invention permits transferring highly stringent run conditions from
one multicapillary instrument to another, or between multicapillary
and single-capillary instruments.
[0045] Although the specific examples shown here are related to
CDCE, the thermostat array control system of this invention can be
used in any physical, chemical, or bioanalytical application in
which stable, accurate and resettable temperature is critical to
achieving quality results, including mutation discovery by SSCP,
cellular analysis by flow cytometry, instrumentation for
immunoassays (binding assays), automated and inline sample
preparation for hematology and/or immunology, and protein
separations, liquid chromatography including high performance
liquid chromatography, and long-read DNA sequencing.
Implementations of the same approach with miniature heaters
embedded in microfabricated devices can also be contemplated.
Moreover, the method can be extended to the production of temporal
and spatial temperature gradients during separation. For example,
temporal gradients are achieved by ramping the target temperature
in the control software during the separation, and spatial
gradients are achieved by setting different target temperatures for
a plurality of physically separated heating elements located on a
given heater. Temperature cycling, and other temperature profiles
of arbitrary complexity, are also feasible. Further, this heater
system can be used both for CTCE and CDCE within a single
instrument. In one embodiment, one could conduct CTCE runs for a
preliminary separation of heteroduplex peaks and then follow up
with a CDCE run for a higher resolution separation and
analysis.
[0046] While the present invention has been described in
conjunction with a preferred embodiment, one of ordinary skill,
after reading the foregoing specification, will be able to effect
various changes, substitutions of equivalents, and other
alterations to the compositions and methods set forth herein. It is
therefore intended that the protection granted by Letters Patent
hereon be limited only by the definitions contained in the appended
claims and equivalents thereof.
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